RNA-seq analysis workflow

Preparation

Load packages

library(dplyr)
library(ggplot2)
library(DESeq2)
library(edgeR)
library(reshape)
library(dplyr)

Sample data

Data import (count matrix).
Data import (TPM).

TPM calculation

Calculate TPM sample by sample

for(i in 1:ncol(count_mtx)){   
  rpkm = count_mtx[,i]/(gene_length/1000)   
  rpkm_sum = sum(rpkm)   
  tpm <- rpkm / rpkm_sum * 1e6   
  tpm_all[,i] = tpm   
}

Principal component analysis

ggplot(pcaData, aes(x = PC1, y = PC2, fill = group)) +
  geom_point(size = 4, alpha = 0.6, shape = 21, color = "black", stroke = 0.5)  +
  ggrepel::geom_text_repel(aes(label=name), 
                           color="grey6", size=3, hjust= -0.3, vjust=-0.3) +
  labs(x = paste("PC1: ", round(100 * PCA_var[1]), "% variance"),
       y = paste("PC2: ", round(100 * PCA_var[2]), "% variance")) +
  theme_bw() +
  theme(legend.title = element_blank()) +
  ggtitle("PCA") +
  labs(caption = " ")

Differentially Expressed Genes (DEG) analysis

DEG data

# Generate info table
info <- data.frame(matrix(nrow = ncol(count.mtx), ncol = 2))
colnames(info) <- c('sample', 'cond')
info$sample <- colnames(count.mtx)
info$cond <- "sample information here"

# DESeq
dds <- DESeqDataSetFromMatrix(count.mtx, info, ~ cond)
dds <- DESeq(dds)
res <- results(dds)

Visualization of DEGs Volcanoplot

res = res %>% mutate(DE=ifelse(log2FoldChange >= log2(fc) & padj < pval, 'UP',
                               ifelse(log2FoldChange <= -log2(fc) & padj < pval, 'DN','no_sig')))
res$DE = factor(res$DE, levels = c('UP','DN','no_sig'))

res %>% 
  ggplot(aes(log2FoldChange, -log10(padj), color=DE)) + 
  geom_point(size=1, alpha=0.5) + 
  scale_color_manual(values = c('red','blue','grey')) +
  theme_classic() +
  geom_vline(xintercept = c(-log2(fc),log2(fc)), color='grey') +
  geom_hline(yintercept = -log10(0.05),color='grey') +
  guides(colour = guide_legend(override.aes = list(size=5))) +
  ggtitle("Resist/Naive") +
  ggeasy::easy_center_title() ## to center title

Gene set enrichment analysis

Perform GSEA

library(clusterProfiler)
gobp <- msigdbr::msigdbr(species = "Mus musculus", 
                         category = "C5", subcategory = "GO:BP") %>% 
  dplyr::select(gs_name, gene_symbol)

perform_GSEA <- function(res, ref, pvalueCutoff = 1) {
  ranking <- function(res) {
    df <- res$avg_log2FC
    names(df) <- rownames(res)
    df <- sort(df, decreasing = TRUE)
    return(df)
  }
  
  ranked.res <- ranking(res)
  
  x <- clusterProfiler::GSEA(geneList = ranked.res,
                             TERM2GENE = ref,
                             pvalueCutoff = pvalueCutoff,
                             pAdjustMethod = "BH",
                             verbose = TRUE,
                             seed = TRUE)
  
  result <- x@result %>% arrange(desc(NES))
  result <- result[, c('NES', 'pvalue', 'p.adjust', 'core_enrichment', 'ID')]
  return(result)
}

# Application 
gsea.res = perform_GSEA(res = deg.cluster3, ref = gpbp)

NES plot

# GESA Plot 
gsea_nes_plot =function(gsea.res, title){
  gsea.res %>% ggplot(aes(reorder(ID, NES), NES)) +
    geom_col(aes(fill=p.adjust)) +
    coord_flip() +
    labs(x="Pathway", y="Normalized Enrichment Score",
         title= "GSEA") + 
    theme_classic() +
    scale_fill_gradient(low = 'red', high = '#E5E7E9') +
    theme(axis.text.x= element_text(size=5, face = 'bold'),
          axis.text.y= element_text(size=6, face = 'bold'), 
          axis.title =element_text(size=10)) +ggtitle(title)
}

# Application 
gsea_nes_plot(gsea.res[gsea.res$p.adjust <= 0.05,], title = "Cluster3")

Enrichment plot for the individual pathway

id1 = "GOBP_NUCLEOSOME_POSITIONING 
enrichplot::gseaplot2(gsea.res, geneSetID = id1, title = id1)

ssGSEA

Single sample GSEA

Single-sample Gene Set Enrichment Analysis (ssGSEA) is an variation of the GSEA algorithm that instead of calculating enrichment scores for groups of samples (i.e Control vs Disease) and sets of genes (i.e pathways), it provides a score for each each sample and gene set pair.
(https://www.genepattern.org/modules/docs/ssGSEAProjection/4#gsc.tab=0)

tpm = read.csv("../sampledata/tpm.sample_mouse.csv", row.names = 1)

## Perform ssgsea 
library(corto)

## Input data : tpm (count.mtx is accepted as well) 
test = ssgsea(tpm,hallmarkList)

## Reshape it to plot 
test.df = test %>% t() %>% data.frame()
rownames(test.df) = colnames(tpm)

## p value of ssgsea 
pval = corto::z2p(test)
colnames(pval) = rownames(test.df)

ssGSEA heatmap

Color represents enrichment score

## Heatmap 
my.color=c(colorRampPalette(colors = c("#2874A6","white"))(60),
           colorRampPalette(colors = c("white","#D35400"))(70))
t(test.df) %>% pheatmap::pheatmap(color = my.color, 
                                  cluster_rows = F,
                                  cluster_cols = F,
                                  main = "ssGSEA of HALLMARK pathways",
                                  gaps_col = c(3,6)) # Simple heatmap

Significance by p value Heatmap

Red: p value <= 0.05 (significant)
White : p value > 0.05 (not significant)

## Heatmap 
input.data = (pval<=0.05)
input.data[] <- +input.data
input.data %>% pheatmap::pheatmap(color = c("grey99","salmon"), 
                                  cluster_rows = F,
                                  cluster_cols = F, 
                                  main = "Significance by p value",
                                  gaps_col = c(3,6)) # Simple heatmap

Single pathway ssGSEA

Pathway sample 1

# Plot it 
genesetname = "WP Apoptosis"
test.df = test.df %>% 
  mutate(significance = ifelse(TCSA_pval <= 0.05, max(test.df[,1]),0))
input = data.frame(test.df[,c(1,3)])
rownames(input) = rownames(test.df)
colnames(input) = c(genesetname, "p value < = 0.05")
my.color=c(colorRampPalette(colors = c("#2874A6","white"))(50),
           colorRampPalette(colors = c("white","#D35400"))(40))
input %>% pheatmap::pheatmap(cluster_rows = F, cluster_cols = F, 
                             color=my.color, border_color = "grey33", 
                             gaps_col = 1, 
                             main = "ssGSEA with significance")

Pathway sample 2

# Plot it 
genesetname = "GPBP DNA Damage"
test.df = test.df %>% 
  mutate(significance = ifelse(TCSA_pval <= 0.05, max(test.df[,1]),0))
input = data.frame(test.df[,c(1,3)])
rownames(input) = rownames(test.df)
colnames(input) = c(genesetname, "p value < = 0.05")
my.color=c(colorRampPalette(colors = c("#2874A6","white"))(50),
           colorRampPalette(colors = c("white","#D35400"))(60))
input %>% pheatmap::pheatmap(cluster_rows = F, cluster_cols = F, 
                             color=my.color, border_color = "grey33", 
                             gaps_col = 1, 
                             main = "ssGSEA with significance")

Multiple genes-violin plot across samples

# Import sample data 
tpm.sample = read.csv("../sampledata/tpm.sample.csv", row.names = 1)

cellmarker = read.csv('../info/DB/CellMarker2.0.csv')
celltypes = c("B cell",
              "CD8+ T cell",
              "Activated T cell",
              "Cancer cell",
              "CD4+ T cell",
              "Dendritic cell",
              "Macrophage")

# Functions  
mks.violinplot = function(cellname, tpm=tpm.sample){
  rs <- grepl(cellname, cellmarker$cell_name, ignore.case = TRUE)
  
  mks = cellmarker[rs, ]$marker 
  mks = mks[mks %in% rownames(tpm)]
  
  tpm.df =tpm %>% filter(rownames(.) %in% mks)
  tpm.df$gene = rownames(tpm.df)
  
  p= tpm.df %>% reshape::melt() %>% ggplot(aes(.[,2], log10(.[,3]))) +
    
    geom_violin(aes(fill = variable), alpha = 0.1, show.legend = FALSE) +
    geom_boxplot(outlier.size = 0, width = 0.2, alpha = 0.5, show.legend = FALSE) +
    geom_jitter(aes(color = variable), alpha = 0.5, size = 1, show.legend = FALSE) +
    scale_y_continuous(labels = scales::comma) +
    theme_bw() +
    labs(y = paste0(cellname, "  genes (log10 scaled)"),
         title = paste0(cellname, " signature genes ")) +
    theme(
      axis.title.x = element_blank(),
      axis.text.x = element_text(angle = 90, hjust=1, size=14),
      panel.grid.major.x = element_blank(),
      axis.title.y = element_text(size = 14),  # y 축 레이블 폰트 크기 설정
      plot.title = element_text(size = 20)
    )
  return(p)
}

mks.violinplot(cellname = "B cell", tpm=tpm.sample)

mks.violinplot(cellname = "Dendritic cell", tpm=tpm.sample)

More example analyses will be updated.